The overall objective of this research is to obtain and characterize the entire bovine elastin gene including the 5' flanking region and to study regulation of elastin gene transcription. We have already obtained and sequenced a 1300 bp cDNA from sheep elastin and characterized 10 kb of the 3' end of the bovine elastin gene. The remainder of the bovine elastin gene will be obtained by screening of lambda and cosmid genomic libraries. Once obtained, the gene will be characterized by restriction enzymes, S1 mapping and DNA sequencing. Detailed characterization of the elastin gene is important to: (1) determine the primary structure of elastin which has not been completely determined at protein level, (2) find out whether the elastin gene arose from gene duplication of the hydrophobic and crosslink regions, (3) identify possible multiple types of elastin. Bovine nuchal ligament fibroblasts will be utilized to study the mechanisms involved in the control of elastin synthesis. Previous observations on transcriptional control of elastin synthesis will be extended by using cDNA probes to quantitate elastin mRNA extracted from nuchal ligaments of various age bovine foetuses and from cultured fibroblasts derived from these ligaments. The mechanism of the glucocorticoids stimulation of elastin synthesis will be investigated by similar method. Our final aim will be to determine the elements in the elastin gene which are responsible for control of transcription. The gene for an easily assayable enzyme, chloramphenicol acetyltransferase (CAT) will be fused to the elastin promoter in a recombinant plasmids. The transfected recombinant plasmids will be used to study transcriptional control in (1) bovine nuchal ligament fibroblasts from various age foetuses which normally synthesize elastin to varying degrees, (2) bovine tendon fibroblasts which normally do not synthesize elastin, (3) bovine nuchal ligament fibroblast treated with glucocorticoids. The amount of endogenous mRNA obtained from nuchal ligament fibroblast will be quantitated and compared with the CAT activity in each experiment. Finally, the CAT assay will be used to identity possible enhancer elements contained in the elastin gene. The elastin fiber is an important connective tissue element in the cardiovascular system, in the lung, in the dermis and other specialized structures. A clear understanding of elastin structure and control of elastin synthesis is fundamental to understanding the development of these tissues and their involvement in genetic and acquired diseases.